Dioxetane labeled probes and detection assays employing the same

Abstract

Probes labeled with 1,2-dioxetane precursors can be employed in a variety of assays. The probes may be nucleic acid, peptide nucleic acid, proteins including enzyme, antibody or antigen, steroid, carbohydrate, drug or non-drug hapten. The probe is provided with a 1,2-dioxetane precursor bound thereto, generally either covalently, or a strong ligand bond. The dioxetane precursor moiety is converted to a bound 1,2-dioxetane by exposure to singlet oxygen. These dioxetane (labels) either spontaneously decompose, or are induced to decompose by an appropriate trigger to release light. The trigger may be a change in pH temperature, or an agent which removes a protective group. Assay formats in which these 1,2-dioxetane labeled probes and referents may be used to include hybridization assays, immuno assays, gel-based assays and Capillary Zone Electrophoresis.

What is claimed is:  
     
       1. A labeled biological probe, comprising: 
       a first biological moiety selected from the group consisting of a nucleic acid, a peptide nucleic acid, a protein, a steroid and a carbohydrate, said biological moiety bearing a 1,2-dioxetane precursor bound thereto, such that, upon exposure to singlet oxygen, said precursor is converted to a 1,2-dioxetane moiety bound to said biological moiety which 1,2-dioxetane moiety subsequently decomposes to release light, wherein the 1,2-dioxetane moiety has the formula:                    
        wherein T is a polycycloalkyl group, R is a substituted or unsubstituted alkyl, cycloalkyl or aryl group, Ar is an aryl moiety, X is an enzyme-cleavable group and Y is hydrogen or other electron donating or electron withdrawing group.  
     
     
       2. A labeled non-biological probe, comprising: 
       a first non-biological moiety selected from the group consisting of a pharmaceutical drug, a non-pharmaceutical drug and a non-drug hapten, said non-biological moiety bearing a 1,2-dioxetane precursor bound thereto, such that, upon exposure to singlet oxygen, said precursor is converted to a 1,2-dioxetane moiety bound to said biological moiety, which 1,2-dioxetane moiety subsequently decomposes to release light, wherein the 1,2-dioxetan moiety has the general formula:                    
        wherein T is a polycycloalkyl group, R is a substituted or unsubstituted alkyl, cycloalkyl or aryl group, Ar is an aryl moiety, X is an enzyme-cleavable group and Y is hydrogen or other electron donating or electron withdrawing group.  
     
     
       3. An assay method to detect the presence of a target in a sample, comprising: 
       combining the labeled probe of  claim 1  with said sample under conditions which promote the formation of a single chemical entity in which said probe and any said target present in said sample are bound,  
       exposing said bound probe to singlet oxygen to form said 1,2-dioxetane moiety, and  
       detecting light emitted by said dioxetane upon decomposition thereof, wherein said light emitted is indicative of the presence and amount of said target.  
     
     
       4. A method of conducting a gel migration assay for analysis, comprising: 
       combining a labeled probe of  claim 1  or  2  specific for a target with a sample to be inspected for the presence of said target,  
       distributing said combined sample and target on a migration gel,  
       inducing separate components of said sample and said probe to differentially migrate across said gel,  
       exposing said probe to singlet oxygen to form said 1,2-dioxetane moiety, and  
       inducing said 1,2-dioxetane moiety to decompose and release light, wherein said light release is monitored, and that portion of the gel wherein light release is observed is identified, and any said target will be present in said identified gel.  
     
     
       5. A method of conducting a size base sequence analysis of a nucleic acid sequence, comprising: 
       enzymatically digesting said sequence to obtain nucleic acid sequence fragments, functionalizing a terminal end of each said fragment so as to bind a 1,2-dioxetane precursor moiety thereto,  
       causing said functionalized fragments to differentially migrate across a gel, and  
       exposing said precursor moieties to singlet oxygen so as to convert said precursor moieties to 1,2-dioxetane moieties bound to said fragments, causing said 1,2-dioxetanes to decompose and chemiluminesce, wherein said chemiluminescence is monitored, and each portion of said gel wherein chemiluminescence is observed contains a fragment which may be eluted therefrom, and wherein the 1,2-dioxetane moiety has the general formula:                    
        wherein T is a polycycloalkyl group, R is a substituted or unsubstituted alkyl, cycloalkyl or aryl group, Ar is an aryl moiety, X is an enzyme-cleavable group and Y is hydrogen or other electron donating or electron withdrawing group.  
     
     
       6. A labeled probe, comprising: 
       a first moiety selected from the group consisting of a nucleic acid, peptide nucleic acid, protein, steroid, carbohydrate, pharmaceutical drug, non-pharmaceutical drug, and a non-drug hapten, wherein said first moiety corresponds to a target component of a sample, said first moiety bearing bound to it a 1,2-dioxetane moiety which, upon exposure to a suitable trigger, decomposes to release light, wherein the 1,2-dioxetane moiety has the general formula:                    
        wherein T is a polycycloalkyl group, R is a substituted or unsubstituted alkyl, cycloalkyl or aryl group, Ar is an aryl moiety, X is an enzyme-cleavable group and Y is hydrogen or other electron donating or electron withdrawing group.